Method of producing antibodies to a restricted population of T lymphocytes, antibodies produced therefrom and methods of use thereof

ABSTRACT

This invention relates to a method of obtaining antibodies specific to a population of T-lymphocytes (T cells) restricted by Vβ gene usage. The invention also relates to the antibodies obtained and to methods of using them. According to the invention, T cells are incubated with an effective amount of a superantigen under conditions and for a time sufficient to allow division and growth of T-cells reactive to the superantigen. The incubated T-cells are injected into a mammal, and thereafter serum containing antibodies specific to the incubated and injected T-cells is removed from the mammal.

This invention was made in part with support under grants CA-49283,AI-28367, AI-31140, CA-42046, AR-02255, AI-12103 and P60-AR-38520awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

This invention relates to a method of obtaining antibodies specific to apopulation of T lymphocytes (T cells, TCL) restricted by Vβ gene usage,antibodies thus obtained and methods of use thereof.

More than 95% of all T cells have a cell surface receptor designated the"T-cell receptor" (hereinafter, "TCR"). For a review of T cell ontogeny,see Moyer and Reinherz, "T Lymphocytes: Ontogeny, Function, andRelevance to Clinical Disorders", N. Engl. J. Med., 317:1136-1142(1987). TCR consists of a clonotypic Ti α-β heterodimer with an apparentmolecular weight (Mw) of 90 kD and a monomorphic T3 molecule containingfive subunits (γ, δ, ζ, ε, and η). The γ subunit has an apparent Mw of25 kD, the δ and ε subunits each have an apparent Mw of 20 kD, the ζsubunit has an apparent Mw of 16 kD, and the η subunit an apparent Mw of22 kD. All five receptor subunits are transmembrane proteins. As is thecase with antigen-binding heavy and light chains of immunoglobulinproteins, TCR α and β proteins contain both variable (hereinafter "V")and constant (hereinafter "C") regions. For a review of immunoglobulinproteins and gene usage, see Lewin, Genes III, John Wiley & Sons, Inc.pp. 642-653 (1987), which is incorporated herein by reference.

It appears that the Ti subunits form a binding site for antigen and themajor histocompatibility complex (hereinafter "MHC") through interactionof their V domains. Antigen recognition is important for activation ofboth cytotoxic effector T cells and immunoregulatory T cells. CytotoxicT cells lyse specific target cells, including tumor cells andvirus-infected cells, whereas immunoregulatory T cells induce orsuppress the cells of the immune system either directly or indirectlythrough lymphokines.

Analyses of many of the cDNA nucleotide sequences encoding a variety ofβ chains have led to the recognition of structural similarity betweengenes encoding β chains and genes encoding immunoglobulins. Thus, genesencoding β chains contain V, constant (C), joining (J) and diversity(D)-like elements The present invention is concerned with the V regionof the chain, hereinafter termed Vβ. In humans, approximately 57 Vβgenes are known to exist in the Ti β locus on chromosome 7 at 7q35Robinson, "The Human T Cell Receptor β-chain Gene Complex Contains atLeast 57 Variable Gene Segments", J Immunol , 146:4392-4397 (1991)

T cell proliferation requires the interaction of the Ti complex withantigen and interleukin-2 (hereinafter "IL-2"). Although resting T cellsexpress no receptors for IL-2; after the T cell receptors are activatedby antigen and MHC, induction of IL-2 receptors occurs within hours Theactivation also leads to endogenous induction and secretion of IL-2, DNAsynthesis and cell mitosis.

Various disease states and physiological disorders are associated with Tcell dysfunctions. These disorders, are characterized by a particularsubset, or restricted population, of T cells which are thought to beresponsible for the dysfunction The restricted population is recognizedby its expression of only one or a few related types of TCR and can bemonitored by the type of Vα and/or Vβ gene expressed. A restricted setof T cells is one in which the T cells express one or a few common Vgenes but are otherwise dissimilar By comparison, a clonal population ofT cells, such as may be derived from a tumor, is a population of T cellsthat are the progeny of a single cell and are hence virtually identical.

Disorders thought to be caused by T cell dysfunctions include but arenot limited to various autoimmune diseases such as systemic lupuserythematosus multiple sclerosis, myasthenia gravis, diabetes mellitus,and various forms of arthritis such as rheumatoid arthritis. Thesedysfunctions are characterized by the expansion of a restricted T cellpopulation that expresses one or a few Vβ genes from a Vβ family.Different patients express a single Vβ but not necessarily the same Vβgene as another patient. For instance, expanded T cell populations fromMS patients express one of the V genes from the related group of Vβ12,Vβ13, Vβ14, Vβ15 and Vβ17. Several methods of therapy have been proposedbased on eliminating or blocking the T cell population responsible for adysfunction. For review see Janeway, "Immunotherapy by Peptides" Nature341:482-483 (1989); and Hashim et al., "Antibodies for Vβ8 ReceptorPeptide Suppress Experimental Autoimmune Encephalomyelitis", J.Immunol., 144:4621-4627 (1990), which are incorporated herein byreference.

There is considerable evidence of selective TCR Vβ gene usage amongrodent T cells which mediate a number of experimental autoimmunediseases. For example, in experimental allergic encephalomyelitis (EAE),Vβ8.2⁺ T cells play a central role. In five different strains of rats,encephalitogenic T cell clones and hybridomas, reactive against myelinbasic protein (MBP) peptide fragments, were found to be uniformlyVβ8.2⁺. Burns et al., "Both Rat and Mouse TcRs Specific for theEncephalitogenic Determinant of MBP use Similar Vα and Vβ Chain GenesEven Though the MHC and Encephalitogenic Determinants Being Recognizedare Different", J. Exp. Med., 169:27 (1989). Similarly, Vβ8.2 isexpressed on over 85% of T cells reacting to the encephalitogenic MBPpeptide in strains of mice susceptible to EAE. Acha-Orbea et al.,"Limited Heterogeneity of T Cell Receptors From Lymphocytes MediatingAutoimmune Encephalomyelitis Allows Specific Immune Intervention" Cell54:563 (1988); and Urban et al , "Restricted Use of T Cell ReceptorGenes in Murine Autoimmune Encephalomyelitis Raises Possibilities forAntibody Therapy", Cell, 54:577 (1988). The mouse Vβ8 gene familyproducts are homologous to the human Vβ12, Vβ13, Vβ14, Vβ15 and Vβ17gene products.

The in vivo administration of mAb specific for Vβ8.2 has been shown toboth protect mice from the development of EAE induced by a subsequentchallenge with MBP, and to ameliorate the clinical course of EAE in micealready affected. Acha-Orbea et al. (1988). It has also been found thatEAE can be vaccinated against. In this case, the anti-T cell response ismediated by another set of T cells Lohse et al,. "Control ofExperimental Autoimmune Encephalomyelitis by T Cells Responding toActivated T Cells", Science, 244:820-824 (1989).

In another animal disease model, collagen-induced arthritis, T cellsreactive against type II collagen which are capable of transferringarthritis to naive syngeneic mice are virtually all Vβ8.2⁺ Banerjee etal., "Possible Role of Vβ T Cell Receptor Genes in Susceptibility toCollagen-Induced Arthritis in Mice", J Exp Med , 167:832 (1988). Theseobservations suggest that expression of the Vβ8.2 gene product may beassociated with an autoimmune T cell pool in rodents, for instance, Tcells derived from the CD4⁻ CD8⁻ Vβ8.2 expressing thymocytesubpopulations. Fowlkes et al., "A Novel Population of T Cell Receptorαβ Bearing Thymocytes Which Predominantly Express a Single Vβ GeneFamily", Nature, 329:251 (1987); Shortman et al., "Mouse StrainDifferences in Subset Distribution and T Cell Antigen ReceptorExpression Among CD4⁻ CD8⁻ Thymocytes", Immunol. Cell Biol., 66:423(1988); and Takahama et al., "Phenotype, Ontogeny, and Repertoire ofCD4⁻ CD8⁻ T Cell Receptor αβ⁺ Thymocytes: Variable Influence ofSelf-Antigens on T Cell Receptor Vβ Usage", J. Immunol., 146:1134(1991).

Recently, it has been possible to determine the TCR gene usage of apopulation of T cells. Bertness et al., "T cell Receptor GeneRearrangements as Clinical Markers of Human T cell Lymphomas", N. Engl.J. Med., 313:534-538 (1985). Such determinations have relied onanticlonotypic antibodies directed at epitopes found on V domains of TCRand cDNA probes that detect clone-specific DNA rearrangements. However,the availability of anticlonotypic antibodies and cDNA probes has beenlimited to the availability of naturally occurring clonal populations ofT cells such as from tumors. This drawback renders these methods lessclinically applicable than would be the case if a wide variety ofantibodies were available to the full range of TCR V gene productsassociated with T cell dysfunctions. This is particularly important in Tcell dysfunctions which are characterized by restricted rather thanclonal populations of T cells. It would be useful to have a method ofobtaining antibodies specific for the protein products of the TCR V genefamilies for the purposes of diagnoses and therapeutics of variousdisorders related to T cell dysfunctions.

SUMMARY OF THE INVENTION

It has now been found that antibodies specific for a restricted set of Tcells having common TCR Vβ gene usage can be obtained by incubating Tcells with an effective amount of a superantigen (SA) under conditionsand for a time sufficient to allow division and growth of T cellsreactive to the SA, injecting the incubated T cells into a mammal andobtaining the anti-Vβ antibodies from the mammal.

Additional antibodies can be made to T cells expressing other TCR Vβgenes in the restricted set, by selectively removing, or depleting, Tcells recognized by an antibody produced by the method of the presentinvention. The remaining T cells are injected into a mammal to produceantibodies. The depletion step may be followed by another cycle of SAexpansion. Depletion, expansion and injection can be repeated a numberof times with a single sample of T cells to provide a panel ofantibodies specific for a variety of Vβ gene products each of whichrecognizes a single restricted set of T cells. Such antibodies andmethods for their use are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph depicting B cell differentiation in response to Tcells exposed to various SA, or media alone. Differentiation isdetermined by IgM production.

FIG. 2A is a bar graph depicting B cell CD23 expression in response to Tcells exposed to the superantigen MAM.

FIG. 2B is a bar graph depicting B cell CD23 expression in response to Tcells exposed to the superantigen SEE.

FIG. 3A is a line graph depicting control immunofluorescence ofperipheral blood lymphocytes after incubation with PBS andfluoresceinated goat antimouse antibody.

FIG. 3B is a line graph depicting immunofluorescence of peripheral bloodlymphocytes after staining with monoclonal antibody CI followed byfluoresceinated goat anti-mouse antibody.

FIG. 3C is a line graph depicting immunofluorescence of SEE-reactive Tcells after staining with monoclonal antibody Cl followed byfluoresceinated goat anti-mouse antibody.

FIG. 3D is a line graph depicting immunofluorescence of MAM-reactive Tcells after staining with monoclonal antibody Cl followed byfluoresceinated goat anti-mouse antibody.

FIG. 4A is a line graph depicting ⁵¹ Cr release by control 8866 B cellslysed by activated T cells.

FIG. 4B is a line graph depicting ⁵¹ Cr release by MAM-treated 8866 Bcells lysed by activated T cells.

FIG. 4C is a line graph depicting ⁵¹ Cr release by TSST-treated 8866 Bcells lysed by activated T cells.

FIG. 5A is a line graph depicting ₅ Cr release by control 8866 B cellslysed by activated Cl+ T cells.

FIG. 5B is a line graph depicting ⁵¹ Cr release by MAM-treated 8866 Bcells lysed by activated Cl+ T cells.

FIG. 5C is a line graph depicting 51Cr release by TSST-treated 8866 Bcells lysed by activated CL+ T cells.

DETAILED DESCRIPTION OF THE INVENTION

SAs are a group of proteins that activate a large proportion of the Tcell repertoire based on dual avidity for MHC class II antigens and TCRepitopes common to products of one or several TCR β chain V genefamilies. Several SA induce massive T cell proliferation and cytokinesecretion and have been implicated in clinical syndromes, frequentlythose characterized by shock and generalized immunosuppression. SAactivation of a more restricted T cell response may also have an effecton the immune system related to autoimmune disorders. Friedman et al.,"A Potential Role for Microbial Superantigens in the Pathogenesis ofSystemic Autoimmune Disease", Arth & Rheum , 34:468-480 (1991).

SA identified to date include microbial and viral proteins. Themicrobial SA include several staphyloccal enterotoxins, a fragment ofthe group a streptococcus M protein, and MAM, a soluble mitogen producedby Mycoplasma arthritidis. Unite et al., "The V specific SuperantigenStaphylococcal Enterotoxin B: Stimulation of Mature T cells and ClonalDeletion in Neonatal Mice", Cell 56:27-35 (1989); Tomai et al.,"Superantigenicity of Streptococcal M Protein", J. Exp. Med.,172:359-362 (1990); and Atkin et al., "Stimulation of Mouse Lymphocytesby a Mitogen Derived from Mycoplasma arthritidisV. A Small Basic Proteinfrom Culture Supernatants is a Potent T cell Mitogen", J. Immunol.,137:1581-1589 (1986) which are incorporated herein by reference. Notethat M. arthritidis is a causative agent of inflammatory arthritis inrodents. Cole and Ward, "Mycoplasma as Arthritogenic Agents", TheMycoplasmas, Vol. IV, New York, Academic Press (1979). The microbialtoxins that function as SA are among the most potent mitogens known.MAM, for example, induces half-maximal T cell proliferation atconcentrations of less than 1×10⁻¹¹ M. Atkin et al. (1986). Virallyencoded SA are typified by those encoded by the mouse mammary tumorvirus. Choi et al., "A Superantigen Encoding in the Open Reading frameof the 3' Long Terminal Repeat of Mouse Mammary Tumour Virus", Nature,350:203-207 (1991).

In the mouse, MAM has been shown to behave as a classic microbial SA,selectively inducing the proliferation of Vβ8⁺ and Vβ6⁺ murine T Cells.Cole et al., "Stimulation of Mouse Lymphocytes by a Mitogen Derived FromM. arthritides. VII. Responsiveness is Associated With Expression of aProduct(s) of the Vβ8 Gene Family Present on the T cell Receptor α/β forAntigen", J. Immunol., 142:4131 (1989). While MAM is mitogenic for humanT cells, the level of proliferation induced is quite modest compared tothat triggered by the S. aureus-derived SA, and no data exist regardingTCR V gene dependence of MAM recognition. It has now been found thatMAM-reactive human T cells utilize a restricted group of TCR Vβ geneproducts. Monoclonal antibody (mAb) C1, the mAb described herein whichwas generated by the immunization of mice with a non-clonal MAM-reactivehuman T cell line, recognizes a disulfide linked heterodimer, consistentwith the α/β TCR, on approximately 3-6% of peripheral T cells, and40-60% of MAM reactive T cells.

As with other mAb specific for TCR V gene products, C1 reacts with asmall fraction of both the CD4⁺ and CD8⁺ subsets of all donors tested,including cord blood T cells. With respect to SA recognition, it wasdetermined that MAM-reactive TCL are greatly enriched in C1⁺ cells,while TCL responsive to SA with which MAM-reactive TCL are lesscrossreactive, SEE and TSST-1, are depleted of C1⁺ T cells. Studiesemploying the polymerase chain reaction to amplify TCR β chain cDNA fromC1⁺ TCL cells demonstrate that C1 identifies an epitope expressed on theVβ17 gene product. Taken together, these results show that MAMrecognition by human T cells is restricted by TCR Vβ gene usage and thata major fraction of MAM-reactive human T cells are Vβ17⁺.

The present invention thus includes antibodies produced by the methodsdescribed herein. In particular, mAb C1 is encompassed by the presentinvention. mAb C1 has been deposited in the American Type CultureCollection (ATCC) and been given accession HB 10874.

It has now been found that mAb C1 is a unique example of a TCR V genespecific mAb generated by immunizing with a restricted T cellpopulation. As discussed above, MAM is a relatively weak mitogen forhuman T cells, suggesting a restricted population of MAM-reactive Tcells. MAM is thus the preferred SA for use in the present invention,however, any other SA is embodied by the present invention. Stainingdata demonstrate that the TCR epitope recognized by mAb C1 is expressednot only on >60% of the immunizing TCL, which had been retriggeredrepetitively with MAM, but is also present on a relatively largefraction of peripheral T cells activated by MAM for only several days inprimary short term cultures (Table III). The successful application ofthis method, utilizing TCL responsive to other microbial SA asimmunogens, or MAM reactive T cells depleted of C1⁺ T cells greatlyexpands the available panel of mAbs against human TCR V gene products.

Utilizing mAb C1 to determine what percentage of T cells are recognizedby C1, it was found that on average, C1⁺ T cells represent approximately3-5% of the peripheral T cell pool. The marked enrichment of C1⁺ T cellsin even short-term cultures of MAM activated PBL, provide strong, albeitindirect, evidence that all C1⁺ T cells are MAM reactive.

Additionally, subsets of MAM-reactive T cells express Vβ genes otherthan Vβ17, the population recognized by mAb C1. These other subsets wereshown to exist by several lines of evidence. First, resting peripheralblood T cells, depleted of C1⁺ cells, show an undiminished proliferativeresponse to MAM over a wide range of SA concentrations. Second, only40-60% of T cells which persist in long term cultures maintained byrepetitive retriggering with MAM plus autologous APC are C1⁺. Third, MAMreactive TCL which have been depleted of C1⁺ T cells exhibit potent, SAspecific lysis of MAM bearing target cells. The method described hereincan thus be utilized to identify other Vβ genes used by MAM-reactive Tcells and to generate mAbs against their products. Such a method isfacilitated by depleting the MAM reactive cells of C1⁺ T cells,re-expanding the remaining T cells with MAM and using the resultingcells to induce antibody production. Subsequently produced antibodiescan then be used in repeated rounds of depletion, expansion and antibodyproduction. Depletion of cells recognized by a specific antibody israpid and efficient. For instance, MAM reactive T cells treated with C1,exposed to goat anti-mouse IgG antibodies linked to iron and exposed toa magnet, are selectively depleted of all C1⁺ T cells such that they donot reappear even after repeated rounds of expansion with MAM.

The observation that human MAM-reactive T cells identified by mAb C1 areVβ17⁺ is of particular interest in light of the restricted nature of Tcells in diseases characterized by T cell dysfunctions. Amino acidsequence analysis has demonstrated considerable homology between theproducts of murine Vβ8 genes which are expressed by MAM-reactive murineT cells and several human TCR Vβ gene families, including Vβ17, Vβ12,Vβ13, Vβ14 and Vβ15. Cole et al., "Stimulation of Mouse Lymphocytes by aMitogen Derived from M. arthritidis. VIII. Selective Activation of TCells expressing Distinct Vβ T Cell Receptors From Various Strains ofMice by the `Superantigen` MAM", J. Immunol., 142:4131 (1989), andChothia et al., "The Outline Structure of the T Cell α/β Receptor", EMBOJ., 7:3745 (1988). Moreover, there are reports implicating selectiveusage of Vβ17 and several other TCR Vβ gene families by autoimmune humanT cells. For example, expanded populations of MBP-reactive T cells inthe peripheral blood of multiple sclerosis (MS) patients have beenreported. In this report each individual MS patient used a particularTCR Vβ gene family for TCR in MBP-reactive T cells. Ben-Nun et al.,"Restricted T-cell Receptor Vβ Gene Usage by Myelin BasicProtein-Specific T-cells Clones in Multiple Sclerosis: Predominant GenesVary in Individuals", Proc. Natl. Acad. Sci. USA, 88:2466-2470 (1991).Preferential usage of Vβ17 as well as Vβ12, Vβ14 and Vβ15 among those Tcells reactive against an encephalotogenic MBP peptide presented inassociation with DR2 and DR3, two MHC class II genes that areover-represented in the MS patient population. Wucherpfennig et al.,"Shared Human T Cell Receptor Vβ Usage to Immunodominant Regions ofMyelin Basic Protein", Science 248:1016 (1990) In addition, Vβ17⁺ Tcells have been reported to be enriched among activated T cells isolatedfrom the synovial tissue of patients with rheumatoid arthritis (RA).Howell et al., "Clonal Infiltrates of Activated Vβ17⁺ T Cells inSynovial Tissues of Rheumatoid Arthritis Patients", J. Cell Biochem.Suppl., 15A:295 (1991). Another study shows that Vβ14⁺ T cells areoverrepresented among synovial fluid T cells from patients withrheumatoid arthritis. Pallard and West, Science, 253:325-329 (1991).

According to the method of the present invention, mAb to a non-clonal,but restricted, population of T cells are made as described in detail inthe Examples presented below. Briefly, T cells are isolated from wholeblood or plasma by any method known in the art. Preferred methodsinclude but are not limited to separation of T cells from non-T cells bythe formation of rosettes with SRBC and centrifugation on a FicollHypaque gradient according to standard procedures. The isolated T cellsare then incubated with any SA at a concentration of SA sufficient tocause T cell proliferation. Once the SA is added, the cells express theIL-2 receptor such that exogenous IL-2 must be supplied at some point.Generally, IL-2 is not added initially. This is because a small amountof endogenous Il1-2 is produced and is sufficient to stimulate cellsthat have a strong response to the SA. Exogenous IL-2 is withheld forabout one week to prevent proliferation of cells that respond weakly tothe SA. After SA treatment, the cells are allowed to grow for a suitableamount of time, usually about two to three weeks under suitable growthconditions.

In order to eliminate T cells expressing TCR Vβ gene products, for whichantibodies are already available, the T cells are incubated with ananti-Vβ specific antibody, and subsequently exposed to goat anti-mouseimmunoglobulin anti-body. The immune complexes which form between theanti-mouse antibody, the Vβ specific antibody and the T cells recognizedby the Vβ specific antibody, are then removed, for instance by magneticbeads and a magnet or fluorescence activated cell sorting (FACS)depending on the selective marker attached to the anti-mouse antibody.After allowing the remaining cells to grow, an optional selection stepmay be performed utilizing one of the isolation methods described above.The whole cells are then injected into the animal host under conditionssuitable to cause antibody formation. The antibodies obtained arescreened on the cell line with which the animals were immunized. As anegative control, cells derived from the same donor but treated with adifferent, non-cross reactive SA can be used.

Immunization with the incubated T cells can be effected by methodsincluding but not limited to subcutaneously, intraperitoneally,intravenously, intramuscularly or directly into lymph nodes.

As with all immunogenic compositions for eliciting antibodies, theimmunogenically effective amounts of the T cells must be determinedempirically. Factors to be considered are the immunogenicity of the Tcells, whether or not the T cells will be complexed with or covalentlyattached to an adjuvant or carrier protein or other carrier, route ofadministration, and number of immunizing doses to be administered. Suchfactors are known in the vaccine art and it is well within the skill ofimmunologists to make such determinations without undue experimentation.

The number of T cells needed to stimulate antibody production will varysomewhat according to the nature of the T cells (i.e. which Vβ theyexpress) and animal species, in addition to the factors described above.As little as 1×10⁶ cells may be sufficient to elicit an immune responseand up to about 20-100×10⁶ cells or more could also be used. Preferably,the effective amount to ensure antibody production is about 10×10⁶ cellsfor mice. The T cells are not mixed with an adjuvant or adsorbent.Generally, the cells are merely mixed with a physiologically acceptablecarrier such as normal saline or a buffering compound suitable foradministration to mammals.

The presence of the antibodies of the present invention, eitherpolyclonal or monoclonal, can be determined by various assays. Assaytechniques include but are not limited to immunofluorescence (IF) bycytofluorographic analysis or by cell sorting, indirectimmunofluoroscence, immunoprecipitation, ELISA, agglutination andWestern blot techniques. Analysis of Vβ gene usage can be done by DNAsequencing, preferably a DNA amplification step is added such aspolymerase chain reaction (PCR) as described below.

The preferred technique is IF by cytofluorographic analysis rather thanby cell sorting. Briefly, about 1×10⁵ peripheral blood T cells or SAreactive TCL cells are mixed with hybridoma culture supernatants,washed, counterstained with fluorescein labelled goat anti-mouse IgG,washed and examined for immunofluorescence staining on acytofluorograph, for instance an Ortho IIs. Procedures involving the useof agglutination assays are well known in the art of blood screening.Western blots are performed essentially according to the methodsdescribed by Towbin et al., Proc. Natl. Acad. Sci. USA, 76:4350 (1979).

The antibodies obtained by the method of the present invention can beused in methods of detection of the presence of particular populationsof T cells bearing the gene products of a TCR Vβ chain family and can beused to quantitate the percentage of these populations of T cells in thetotal population of T cells. This is useful in diagnosing variousdiseases related to T cell dysfunctions wherein a particular restrictedpopulation of T cell is over-represented within the entire T cellpopulation. Preferably, the methods which use the antibodies to detectthe presence of particular types of T cells in a sample involvecontacting the sample with at least one of the antibodies underconditions which allow the formation of an immunological complex betweenthe antibody and the specific T cell that may be present in the sample.The formation of an immunological complex if any, indicating thepresence of the specific T cell type in the sample, is then detected andmeasured by suitable means.

Such methods include, but are not limited to, homogeneous andheterogeneous binding immunoassays, such as indirect immunofluorescence,radioimmunoassays (RIA), ELISA and Western blot analyses as discussedabove. The antibodies may be labeled or unlabeled depending on the typeof assay used. Labels which may be coupled to the antibodies are thoseknown in the art and include but are not limited to enzymes,radionucleotides, fluorogenic and chromogenic substrates, cofactors,biotin/avidin, colloidal gold and magnetic particles. Modification ofthe antibodies allows for coupling by any known means to carrierproteins or peptides or to known supports, for example, polystyrene orpolyvinyl microtiter plates, glass tubes or glass beads andchromatographic supports, such as paper, cellulose and cellulosederivatives, and silica.

Preferred assay techniques, especially for large-scale clinicalscreening of patient T cells include but are not limited to indirectimmunofluorescence. For instance, the antibodies may be directly bondedto T cell specimens in solution or in situ in histological specimens anddetected by fluorescence microscopy.

The antibodies are also suitable for use as therapeutic agents. Forinstance, the antibodies may be used unaltered or coupled to toxinsincluding but not limited to ricin and diphtheria toxin and administeredto a patient. Antibodies used alone are capable of fixing complement andinitiating cytolysis of the target cell. Once the antibodies bind to thespecific T cell, they cause the death or removal of the T cell and thusameliorate the dysfunction caused by the T cells. The antibodies aregenerally administered with a pharmaceutically acceptable carrier orvehicle therefor. A pharmaceutically acceptable carrier is one that doesnot cause an adverse physical reaction upon administration and one inwhich the antibodies are sufficiently soluble and retain their activityto deliver a therapeutically effective amount of the compound. Thetherapeutically effective amount and method of administration of theantibodies may vary based on the individual patient, the indicationbeing treated and other criteria evident to one of ordinary skill in theart. A therapeutically effective amount of the antibodies is onesufficient to induce death or removal of a sufficient number of thespecific T cells to ameliorate the dysfunction without causingsignificant side effects such as non-specific T cell lysis or organdamage. The route(s) of administration useful in a particularapplication are apparent to one or ordinary skill in the art.

Routes of administration include, but are not limited to, parenteral,and direct injection into an affected site. Parenteral routes ofadministration include but are not limited to intravenous,intramuscular, intraperitoneal and subcutaneous. For most T celldysfunctions, intravenous administration is preferred, but where thedysfunction is localized, such as in arthritis, direct injection to theaffected site will result in increased effectiveness and decreased sideeffects such as non-specific organ damage.

The invention also encompasses antibodies made in response to the SAreactive T cells and which recognize TCR Vβ proteins. Such antibodiescan be either polyclonal or monoclonal. Methods for making both types ofantibodies are well known in the art. Methods of immunization andantibody production, purification and characterization are known in theart and need not be described in detail. The preferred antibodies aremonoclonal (mAb) and are made by any method known in the art, forinstance by the method described by Kohler and Milstein, "ContinuousCultures of Fused Cells Secreting Antibody of Predefined Specificity",Nature, 256:495-497 (1975) which is incorporated herein by reference.

The present invention includes compositions of the antibodies describedabove, suitable for parenteral administration including, but not limitedto, pharmaceutically acceptable sterile isotonic solutions. Suchsolutions include, but are not limited to, saline and phosphate bufferedsaline for intravenous, intramuscular, intraperitoneal, subcutaneous ordirect injection into a joint or other affected area.

Antibodies used in therapeutics suffer from several drawbacks such as alimited half-life and propensity to elicit an immune response. Severalmethods have been proposed to overcome these drawbacks. Antibodies madeby these methods are encompassed by the present invention and areincluded herein. The use of the words herein "antibodies" and "mAb"include the specific embodiments discussed below. One such method is the"humanizing" of antibodies by cloning the gene segment encoding theantigen binding region of the antibody to the human gene segmentsencoding the remainder of the antibody. Only the binding region of theantibody is thus recognized as foreign and is much less likely to causean immune response. An article describing such antibodies is Reichmannet al., "Reshaping Human Antibodies for Therapy", Nature 332:323-327(1988), which is incorporated herein by reference. Another method toavoid the drawbacks found in antibody therapy can be found in the use ofpeptide analogues which mimic the antigen binding region of the antibodybut are not themselves antibodies. An article describing such antibodymimetics is Saragovi et al., "Design and Synthesis of a Mimetic from anAntibody Complementarity-Determining Region", Science, 253:792-795(1991), which is incorporated herein by reference.

The following Examples are meant to illustrate but not limit the presentinvention.

Example 1 Reagents Used in Succeeding Examples

Staphylococcal enterotoxins SEA, SEB, SEC₁, SEC₂, SEC₃ and SEE as wellas toxic shock syndrome toxin, TSST-1, were obtained from ToxinTechnology, Madison, WI and used according to the manufacturer'sinstructions. Partially purified MAM was isolated from M. arthritidisculture supernatants according to the method described by Atkin et al.,(1986). All SA were used at a final concentration predetermined to beoptimal for T cell proliferation, 1:4000 for MAM and 10-25 ng/ml for theStaphylococcal-derived SA.

Example 2 Isolation and Fractionation of Lymphocytes

Fresh peripheral blood or tonsil lymphocytes were isolated byFicoll-Hypaque centrifugation according to the manufacturer'sinstructions. T cells were isolated from non-T cells by E-rosetteformation with neuraminidase-treated sheep red blood cells according tothe method described by Kaplan and Clark, "An Improved Rosetting Assayfor Detection of Human T Lymphocytes", J. Immunol. Met., 5:131-135(1974), and a second Ficoll-Hypaque centrifugation. Residual T cellswere removed from the non-T cell fraction by treatment with anti-T3antibodies obtained from hybridoma ATCC CRL 8001 (OKT3) obtained fromATCC, followed by the addition of magnetic beads coated with goatanti-mouse antibody and physical separation of the bead-bound T cellsutilizing a magnet according to the manufacturer's instructions (Dynal,Inc., Great Neck, N.Y.).

Example 3 Generation of SA-reactive T helper (T ) cell lines

CD4⁺ peripheral blood T cells were isolated from unselected T cellpopulations obtained as described in Example 2, by incubating the Tcells with an excess of anti-CD8 mAb followed by washing and physicalremoval of T cells binding antibody to CD8 utilizing magnetic beadscoated with goat anti-mouse antibody and a magnet according to themanufacturer's instructions (Dynal). The CD4⁺ -enriched populations werecocultured with X-irradiated autologous antigen presenting cells (APC)and either MAM or SEE. After 5 days, semi-purified human IL-2(Electro-Nucleonics, Inc., Fairfield, N.J.) was added at a finalconcentration of 5%. Cultures were retriggered weekly with APC, and therelevant SA, and expanded in the presence of IL-2. Cell lines weremaintained in culture media consisting of RPMI 1640 (Gibco Laboratories,Grand Island, N.Y.) containing 10% fetal bovine serum (fbs) (Whittaker,Mass. Bioproducts, Walkersville, Md.), penicillin and streptomycin (50μg/ml, Gibco), and 2 mM glutamine (Gibco).

Example 4 CD23 Induction assay

The specific interaction of SA-reactive CD4⁺ human T cells andSA-bearing B cells results in the rapid expression of the CD23activation antigen on a fraction of the resting B cell pool. Friedman etal., "A Potential Role for Microbial Superantigens in the Pathogenesisof Systemic Autoimmune Disease" Arthr & Rheum., 34:468 (1991).

The induction of B cell surface CD23 expression by T_(h) cells has beendetailed previously. Crow et al., (1986). Briefly, 5×10⁵ purifiedtonsillar B cells were cultured in final medium with 1.5×10⁵X-irradiated CD4⁺ MAM- or SEE-reactive TCL cells. Cultures weresupplemented with final medium alone or medium containing an optimalconcentration of the various SA (MAM was used at a 1:4,000 dilution,whereas the other SA were used at 100 ng/ml). After 16 hours, B cellswere assayed for CD23 expression by indirect immunofluorescence stainingutilizing mAb EBVCS₂ (generously donated by Dr. Bill Sugden and StanMetzenberg, Madison, Wis.) and counterstained withfluorescein-conjugated F(ab')² fragments of goat anti-mouse IgGaccording to the manufacturer's instructions (Tago, Inc., Burlingame,Calif.). The percentage of positively staining cells was determined byanalysis on an Ortho IIs cytofluorograph (Ortho Diagnostic Systems,Inc., Westwood, Mass.).

The results obtained show that CD4⁺ SEE-reactive TCL cells induceoptimal CD23 expression on B cells bearing TSST-1 or any of the SEs,but, as shown in Example 7, trigger little CD23 expression by MAMbearing B cells. This functional evidence of crossreactivity bySA-activated TCL cells is consistent with reports that activated human Tcells are somewhat promiscuous in their proliferative responses to thestaphylococcal-derived SA. Fleischer et al. (1991). Importantly,however, the patterns of CD23 expression observed suggest that MAM andSEE-specific human T cells show little cross-reactivity and maytherefore utilize different TCR Vβ gene products. Thus, the SEE-reactiveTCL provide an excellent comparison for screening MAM-specific TCR mAbs.

Example 5 Generation of Monoclonal Antibodies

In order to generate mAb to a clonal population of T cells, Balb/c micewere immunized on 4 occasions with 1×10⁷ MAM-reactive TCL cells in 100μl phosphate buffered saline (PBS, 10 mM NAPO₄, 150 mM NaCl, pH 7.2)which had been expanded in long-term culture (7 weeks) by weeklyrestimulation with autologous APC and MAM as described in Example 2.Essentially, the method described by Kohler and Milstein (1975) was usedto produce the mAb. Briefly, three days after the final immunization,the mice were sacrificed and their splenocytes fused with the HGPRTdeficient myeloma cell line SP2/0 or NS-1. Hybridomas which demonstratedreactivity with a small fraction of freshly isolated (resting)peripheral T cells were screened against the MAM-reactive TCL used forimmunization and an SEE-reactive TCL-derived from the same donor. Thepresence of antibodies was detected by indirect immunofluorescence.Briefly, two color immunofluorescence was prepared by first incubating5×10⁵ cells with various mAb for 30 min. at room temperature. This wasfollowed by 3 washes in PBS-BSA 1%, azide 0.2% and goat anti-mouseIg-FITC (GAM-FITC) for 30 min. at room temperature. The cells werewashed 3 times and incubated with a negative control IgG1 mAb for 30min. at room temperature to quench free GAM-FITC binding sites. Thecells were washed 3 times and incubated with phycoerythrin (PE) labeledanti-CD4 or anti-CD8 mAb (UBI/Olympus, Lake Success, NY) for 30 min. atroom temperature. The cells were finally washed 3 times and analyzed onan ORTHO cytofluorograph. The results shown in Table I represent theratio of double positive (FITC +PE) cells over total CD4 or CD8 positivecells expressed as a percentage. In this manner, a mAb termed Cl, wasidentified.

As shown in FIG. 3, Cl stains between 3-6% of peripheral T cells, >60%of the MAM-reactive TCL used as immunogen, and virtually no SEE-reactiveTCL cells. Immunoprecipitation studies (FIG. 4) showed that mAb C1recognizes a disulfide-linked heterodimer consistent with the α/β TCR.Finally, as with other TCR mAb specific for V gene products, C1recognizes a small subset of peripheral T cells from all donors tested,including samples of cord blood T cells. While C1⁺ cells are found amongboth CD4⁺ and CD8⁺ T cells, some donors show selective enrichment of C1⁺T cells in one or the other T cell subset (Table I).

mAb C1 was utilized to screen a number of SA-reactive TCL propagated invitro. PBL were activated weekly with X-irradiated autologous APC andthe indicated SA. The percentage of T cells staining with each of theanti-TCR mAbs was assessed each week 6 days after retriggering withX-irradiated APCs and SA.

Cord blood lymphocytes and PBL obtained from normal adult donors wereanalyzed by two-color immunofluorescence staining for distribution ofC1⁺ T cells in the CD4⁺ and CD8⁺ T cell subsets. Single colorimmunofluorescence was performed according to the method described byCrow et al. (1986).

                  TABLE I                                                         ______________________________________                                        Percentage of C1.sup.+  T cells in                                            CD4.sup.+  and CD8.sup.+  subpopulations                                                     C1.sup.+  CD4.sup.+                                                                   C1.sup.+  CD8.sup.+                                    ______________________________________                                        Adult blood                                                                              1         14.00     3.77                                                      2         6.40      2.30                                                      3         7.98      5.56                                                      4         5.65      7.87                                                      5         6.63      11.32                                          Cord bloods                                                                              1         7.69      3.76                                                      2         4.75      3.98                                                      3         3.99      3.20                                                      4         3.45      4.48                                                      5         5.65      7.87                                           Mean ± SD         6.62 ± 2.83                                                                          5.41 ± 2.64                                 ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        Peripheral Blood T-cells Triggered                                            With MAM are Highly Enriched in C1                                                           Percentage of T-cells Staining                                 Superantigen   Positively With Anti-TCR mAbs                                  Stimulus       C1      C37    OT145 S511 Ti3a                                 ______________________________________                                        Primary MAM        55.6    0.0  2.5   2.5  5.6                                culture TSST       0.6     1.3  2.8   0.8  1.9                                        SEA        5.1     0.6  2.9   2.9  5.9                                        SEB        26.5    0.4  2.7   9.0  1.5                                        SEE        3.0     3.4  1.2   0.2  12.5                                       SEC1       17.8    4.8  3.0   7.3  5.2                                        SEC2       15.1    1.8  0.0   7.1  1.7                                        SEC3       9.2     4.1  1.9   4.5  6.2                                Secondary                                                                             MAM        39.4    1.2  0.2   2.8  2.7                                culture TSST       0.2     0.0  1.1   0.0  0.0                                        SEA        1.2     0.0  0.0   0.0  0.0                                        SEB        10.1    0.6  2.2   6.4  0.7                                        SEE        0.6     1.2  0.5   0.1  15.3                                       SEC1       9.4     1.0  1.4   2.0  3.1                                        SEC2       15.5    1.6  0.0   5.0  0.4                                        SEC3       6.9     0.0  0.0   0.8  0.0                                Tertiary                                                                              MAM        32.4    0.2  0.0   1.5  2.7                                culture TSST       0.0     0.1  0.3   2.6  1.6                                        SEA        0.7     0.3  0.4   0.7  0.9                                        SEB        7.3     0.5  0.0   3.3  3.3                                        SEE        0.1     0.3  2.1   1.3  11.9                                       SEC1       8.7     0.6  0.7   2.5  0.9                                        SEC2       17.0    0.0  0.0   7.4  0.0                                        SEC3       6.2     0.0  0.1   1.8  0.2                                ______________________________________                                    

As shown (Table II) short term activation of peripheral T cells with apanel of SA demonstrates a clear enrichment of C1⁺ cells among the Tcells activated by MAM and several of the SE with which MAM-specific TCLcells crossreact in the CD23 induction assay, in particular, SEB, SEC1,SEC2, SEC3. In contrast, C1⁺ T cells are not well represented amongnon-crossreactive TSST-1 or SEE-activated T cells. T cells expanded byweekly retriggering with SEB or SEC 1 and autologous APC show a markedfall off in the percentage of C1⁺ T cells (Table II).

These results indicate that SA such as SEB are recognized by T cellsexpressing several TCR Vβ gene family products among which C1⁺ T cellsare a minor component with a relatively low binding affinity for SEB. Incontrast, C1⁺ T cells are greatly expanded in short-term cultures ofMAM-activated T cells and remain well represented. In the experimentpresented in Table 3, (described in Example 8) the percentage of C1⁺ Tcells decreases somewhat over time in the culture stimulated weekly byMAM. However, in most experiments, C1⁺ T cells represent between 50 and60% of TCL repetitively triggered with MAM (FIG. 3). In FIG. 3, PBLshown in the upper right; the CD4⁺ MAM-reactive T cell line used forimmunization shown in the lower right; or a CD4⁺ SEE-reactive T cellderived from the same donor shown in the lower left were analyzed byindirect immunofluorescence staining for reactivity with C1. Backgroundstaining of PBL with PBS and fluoresceinated anti-mouse Ig is shown inthe upper left.

The results obtained indicate that C1⁺ T cells comprise a stablepopulation of 15-20% of SEC₂ reactive TCL cells. These results suggestthat C1⁺ T cells represent the major population of human T cellsreactive with MAM, and a significant fraction of the SEC₂ -responsive Tcell pool.

Example 6 Generation of TCL enriched for TCR V gene usage

Tonsil T cell aliquots were incubated at room temperature withsaturating concentrations of non-cross-reactive TCR V gene specific mAb:C37 (Vβ 5.2/5.3) Wang et al., "A Monoclonal Antibody Detecting a SharedDeterminant on the Human T Cell Antigen Receptor Molecule", Hybridoma,5:179 (1986), OT145 (Vβ 6.7a) Posnett et al., "Inherited Polymorphism ofthe Human T Cell Antigen Receptor Detected by a Monoclonal Antibody"Proc Natl Acad Sci USA, 83:7888 (1986); and Li et al., "AllelicVariations in the Human T Cell Receptor Vβ 6.7 Gene Products", J. Exp.Med., 171:221 (1990) and C1. After 30 minutes, cells were washed 3times, resuspended in final medium and cultured at a final concentrationof 0.5×10⁶ /ml in the presence of goat anti-mouse antibody-coatedmagnetic beads according to the manufacturer's instructions (Dynal).Beads were added at a ratio of 20 beads to 1 target T cell. After 5days, magnetic beads were removed, the T cells washed and reculturedwith IL-2 alone for 48 hours. Cultures were maintained with IL-2 andweekly feeding with periodate-treated allogeneic non-T feeder cells.

These cultures become highly enriched in T cells expressing the relevantVβ gene, depending on the initial mAb used for stimulation. Usually,this occurs over a 6-day period. Occasionally, a second cycle ofstimulation was required to achieve greater than 95% specific Vβexpression. At the time these TCL were utilized as effectors in thecytolytic assay or for RNA isolation as described in Example 10, eachwas virtually 100% for T cells expressing the appropriate TCR Vβ geneproducts.

Example 7 Assay of SA-dependent Cytolysis

In order to show that the MAM-reactive T cell population contains both aC1⁺ and a C1- population of T cells, a functional assay was performed.Anti-TCR mAbs are mitogenic; this characteristic was used to formallyprove that C1⁺ T cells are MAM-reactive by allowing the selectiveactivation and expansion of T cells expressing the relevant TCR epitope.Aliquots of tonsillar T cells were treated with saturatingconcentrations of C1 or either of two non-cross-reactive TCR Vβ geneproduct specific mAb: C37 (Vβ 5.2/5.3, and OT145 (Vβ6.7a). TCL weregenerated as described in Example 2. These lines are virtually pure withrespect to reactivity with the relevant anti-TCR mAb (FIG. 4).

TCL cells were assayed for cytolytic activity in a 4-hour [⁵¹ Cr]release assay according to the method described by Friedman et al.,"Amplification of Altered Self-Reactive Cytolytic T Lymphocyte Responsesby Cloned Allospecific Human T Helper Cells", J. Clin. Invest., 82:1722(1988) which is incorporated herein by reference, utilizing an MHC classII antigen-bearing target cell line, B cell lymphoblastoid cell line8866. Briefly, 8866 cells were incubated for 2 hours at 37° C. with 0.1mCi [⁵¹ Cr] in the presence of final medium alone, or the indicated SA.

Peripheral blood T cells were expanded in culture by weekly activationwith mAb C1, goat anti-mouse Ig-coated magnetic beads, and IL-2 in orderto generate a C1⁺ TCL. In addition, an aliquot of the peripheral T cellpopulation was depleted of C1⁺ cells utilizing the magnetic beads, andactivated with MAM, autologous X-irradiated APC's, and IL-2. ThisMAM-reactive C1⁻ TCL was retreated weekly to insure complete depletionof residual C1⁺ T cells. At the time these TCL cells were used aseffectors in the cytolytic assay (after 4 weeks of culture), less than0.5% of the TCLs stained with C1. Both the MAM-reactive C⁻ TCL and theC1⁺ TCL were utilized as effector cells in a cytolytic assay against [⁵¹Cr]-labeled 8866 target cells, either untreated or "pulsed" with SA asdescribed above. Both TCL efficiently and specifically lyse MAM bearingtarget cells, suggesting a MAM reactive C1⁻ T cell population.

As shown in FIG. 5, both the MAM-reactive TCL and the C1⁺ TCLspecifically and efficiently lyse the MAM bearing 8866 target cells.Data are presented as mean percent lysis of target cells at eacheffector to target cell ratio. These data confirm that C1⁺ T cells areMAM reactive. Taken together, these findings support the existence of aMAM-reactive human T cell population distinct from that which expressesthe C1 epitope.

FIG. 4 shows the results obtained when tonsillar T cells, expanded inculture by weekly activation with an anti-TCR mAb, goat anti-Ig-coatedmagnetic beads, and IL-2, in triplicate at the effector to target ratiosindicated in FIG. 4 for cytotoxic activity against [⁵¹ Cr] releaseassay. Target cells consisted of a lymphoblastoid B cell line 8866either untreated (8866) or "pulsed" for one hour at 37° C. with MAM(8866_(MAM)) or TSST-1 (8866_(TSST-1)). Briefly, purified tonsillar Bcells were cultured with medium alone or the indicated SA at 100 ng/ml,except MAM, which was used at 1/4000 dilution. CD4⁺ MAM-reactive TCLcells or CD4⁺ SEE-reactive TCL cells were added, cultures incubated for16 hours, then analyzed for CD23 expression by indirectimmunofluorescence staining. Depicted are the cytotoxic activities ofC37 activated T cells; mAb 0T145 activated T cells; and C1 activated Tcells. The phenotype of these three cell lines at the time of assay areas follows:

C37 activated=99% C37⁺, 22% CD4⁺, 77% CD8⁺

OT145 activated=100% OT145^(+;) 16% CD4^(+;) 85% CD8^(+;)

C1 activated=99% C1⁺ 43% CD4⁺ 61% CD8⁺

While each line contained a CD4⁺ T cell fraction, CD8⁺ T cellspredominated, comprising 60-80% of the TCL population. A CD8⁺ Tcell-dependent function was therefore assessed, by determining if theseTCL cells could lyse MHC class II positive target cells in a SAdependent manner. In the experiment depicted in FIG. 4, no significantlysis of untreated 8866 target cells by any of the TCL is observed.However, the C1⁺ TCL selectively lyse MAM-bearing 8866 cells, while boththe OT145⁺ and C37⁺ TCL cells effectively lyse TSST-1 bearing, but notMAM-bearing, targets. It should be noted that the proliferative responseof human T cells to TSST-1 is reportedly dominated by the Vβ 2⁺fraction. Choi et al., "Selective Expansion of T Cells Expressing Vβ 2in Toxic Shock Syndrome", J. Exp. Med., 172:981 (1990). The lysis ofTSST-1 bearing targets by Vββ6.7a⁺ and Vβ5.2/5.3⁺ TCL cells thereforerepresents another example of the cross-reactivity of activated T cellresponses to S. aureus-derived SA. Fleischer et al., "An EvolutionarilyConserved Mechanism of T Cell Activation by Microbial Toxins: Evidencefor Different Affinities of T Cell Receptor Toxin Interaction" J.Immunol , 146:11 (1991) .

The experiment depicted in FIG. 4 has been performed on three separateoccasions utilizing C1⁺ TCL independently derived from different donors.In all studies, the results are similar to those shown in FIG. 5. Thus,C1⁺ T cells, activated and expanded with C1, demonstrate functionalspecificity for MAM.

Example 8 Immunoprecipitation of TCR Utilizing C1

A MAM-reactive TCL with 60% C1⁺ T cells was radio-iodinated withlactoperoxidase and peroxide, using 25×10⁶ cells and 2.5 mCi [¹²⁵ ].Cell lysis and immunoprecipitations with SPA-Sepharose and monoclonalantibodies were performed as previously described by Posnett et al., "ANovel Method for Producing Antipeptide Antibodies. Production ofSite-Specific Antibodies to the T Cell Antigen Receptor β Chain", J.Biol. Chem., 263:1719 (1988).

The expansion of C1⁺ cells in short-term cultures of SE-activated Tcells (Table II) indicates that C1⁺ T cells can account for the patternof SA responsiveness associated with MAM-reactive TCL (FIG. 2). However,these data do not rule out the existence of C1⁻ MAM-reactive T cells.Indeed, the observation that repetitive triggering of MAM-reactive Tcells with MAM results in a TCL that is maximally 50-60% C1⁺ providesindirect evidence that a C1⁻ MAM-reactive T cell population exists. Toaddress this point, aliquots of fresh peripheral T cells were depletedof C1⁺, C37⁺ or OT145⁺ T cells by treatment with the relevant mAbfollowed by physical removal of the reactive T cells utilizing magneticbeads bearing goat anti-mouse IgG. Peripheral blood T lymphocytes weredepleted of T cells reacting with anti-TCRmAbs C1 or C37 utilizingmagnetic beads coated with anti-mouse IgG. Untreated or mAb-depleted Tcells were assayed, in triplicate, for proliferative responses againstmedium alone, autologous APC, or the indicated SA in the presence ofautologous APC. The percentage of C1⁺ T cells present in each responderT cell population was detected by immunofluorescence staining. In TableIII experiments 1 and 2 describe the results of separate studiesinvolving two different normal donors.

                                      TABLE III                                   __________________________________________________________________________    C1 Depleted T-cells Proliferate in Response to MAM                                              [.sup.3 H]-Tdr incorporation                                Description       (cpm) induced by:                                           of responder           MAM  SEE  TSST-1                                       population   Media                                                                              APC.sub.xr                                                                         APC.sub.xr                                                                         APC.sub.xr                                                                         APC.sub.xr                                   % C1.sup.+                                                                    __________________________________________________________________________    Exp. 1                                                                             E.sup.+                                                                            3.5                                                                              85   6,681                                                                              37,978                                                                             39,313                                                                             69,033                                            E.sup.+  C1                                                                        0.3                                                                              53   1,159                                                                              24,226                                                                             25,667                                                                             55,629                                            E.sup.+  C37                                                                       2.8                                                                              2,969                                                                              1,837                                                                              25,509                                                                             23,847                                                                             59,855                                       Exp. 2                                                                             E.sup.+                                                                            4.6                                                                              82   1,170                                                                              26,748                                                                             122,655                                                                            54,603                                            E.sup.+  C1                                                                        0.9                                                                              267  1,348                                                                              27,721                                                                             99,509                                                                             46,956                                            E.sup.+  C37                                                                       4.1                                                                              239  1,184                                                                              29,513                                                                             77,742                                                                             58,827                                       __________________________________________________________________________

AS shown in Table III, while this procedure efficiently reduces oreliminates the C1⁺ T cell pool, as detected by immunofluorescencestaining, the proliferative response to MAM was not affected. It shouldbe pointed out that T cell populations depleted of C1⁺ cells maintainstrong proliferative responses over a wide range of MAM concentrations(6 log dilutions). In additional studies, the cytolytic activity of aC1⁺ TCL and a MAM-reactive TCL depleted of C1⁺ cells, both derived fromthe same donor were compared.

Example 9 Analysis of TCR Vβ Gene by Polymerase Chain Reaction (PCR)

Three T cell lines were prepared by stimulating normal peripheral bloodT cells with either OT145 (Vβ6.7), C37 (Vβ5.2/5.3) or C1 mAb asdescribed in Example 8. Total cellular RNA was isolated from each cellline by the acid guanidinium thiocyanate-phenol-chloroform method.Chomczynski and Sacchi, "Single-step Method of RNA Isolation byGuanidinium Thiocyanate-Phenol-Chloroform Extraction", Anal. Biochem.,165:156 (1987).

cDNA was synthesized with reverse transcriptase, using an anti-sense Cβprimer the sequence of which is described below, according to the methoddescribed by Li et al., (1990).

The PCR was performed with a panel of Vβ specific sense primers, inparallel reactions where each Vβ primer was matched with the Cβanti-sense primer situated 55 bp from the 5' end of the C region. Twopaired Cβ primers were used as a positive control. Each cDNA preparationwas tested for the optimal dilution. PCR conditions included primers at0.5 μ, Replinase (Dupont) 2 U, buffer containing 3.0 mM MgCl₂ (20buffer, Dupont), [³² P]-dCTP 20 μCi, cold dNTPs at 0.2 mM each in afinal volume of 20 μl . Amplification was done for 1 min. at 94° C., 1min at 51° C. and 1 min at 72° C. for 25 cycles.

PCR products were analyzed using polyacrylamide gel electrophoresis on a5% polyacrylamide gel. The gel was dried and exposed to film.

    __________________________________________________________________________    Cβ                                                                              (anti-sense)                                                                        5'                                                                              CTTCTGATGGCTCAAACAC                                                                            3' (SEQ ID NO: 1)                             Cα                                                                         5'  (sense)                                                                             5'                                                                              GAACCCTGACCCTGCCGT                                                                             3' (SEQ ID NO: 2)                             Cα                                                                         3'  (anti-sense)                                                                        5'                                                                              TCATAAATTCGGGTAGGATC                                                                           3' (SEQ ID NO: 3)                             Vβ                                                                          2   (sense)                                                                             5'                                                                              GTTTCTCATCAACCATGCAA                                                                           3' (SEQ ID NO: 4)                             Vβ                                                                          6   (sense)                                                                             5'                                                                              TCAGGTGTGATCCAATTTC                                                                            3' (SEQ ID NO: 5)                             Vβ                                                                          5.3/5.2                                                                           (sense)                                                                             5'                                                                              GTCAGGGGCCCCAGTTTAT                                                                            3' (SEQ ID NO: 6)                             Vβ                                                                          17  (sense)                                                                             5'                                                                              ACAGCGTCTCTCGGGAGA                                                                             3' (SEQ ID NO: 7)                             __________________________________________________________________________

Specific PCR amplification of Vβ17 gene products from a C1⁺ TCL wasperformed. The PCR amplified cDNA from three cell lines (OT145⁺, C37⁺,C1⁺) were obtained with Cα primer (positive control), Cβ-Vβ2 primers(negative control), Cβ-Vβ5.2/5.3 primers, Cβ-Vβ6 primers or Cβ-Vβ17primers. specific bands are indicated with arrows. In each case, thebands migrated as expected based on the estimated size of the amplifiedsegment.

TCL were prepared with three mAb: OT145 (Vβ6.7α), C37 (Vβ5.2/5.3), andC1, as described above. Each of these polyclonal T cell linescontained >98% cells positive with the relevant mAb. RNA was isolatedand cDNA synthesized with reverse transcriptase. Aliquots of cDNA werePCR amplified with different primer combinations. The results obtainedshowed that each cell line expressed a specific Vβ . As expected, theOT145⁺ cells expressed Vβ6, and the C37⁺ cells expressed Vβ5.2/5.3. TheC1⁺ cells expressed Vβ17. None of these TCL expressed Vβ2 and all ofthem expressed Cα. In other experiments the C1⁺ cell line was analyzedwith primers specific for Vβ1-Vβ20. No primer combinations other thanVβ17-Cβ amplified a β-chain product. Thus, Vβ17 appears to represent thesole Vβ gene product recognized by C1. Vβ17 is thought to represent a Vβfamily with a single gene copy based on counting bands on Southernblots. Robinson, "The Human T Cell Receptor β Chain Gene ComplexContains at Least 57 Variable Gene Segments: Identification of Six VβGenes in Four New Gene Families", J. Immunol., 146:4392 (1991);Concannon et al., "Diversity and Structure of Human T Cell Receptor βChain Variable Region Genes", Proc. Natl. Acad. Sci. USA, 83:6598(1986); and Kimura et al , "Sequences and Repertoire of the Human T CellReceptor α and β Chain Variable Region Genes in Thymocytes", Eur. J.Immunol., 17:375 (1987).

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 7                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: YES                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CTTCTGATGGCTCAAACAC19                                                         (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GAACCCTGACCCTGCCGT18                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TCATAAATTCGGGTAGGATC20                                                        (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GTTTCTCATCAACCATGCAA 20                                                       (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TCAGGTGTGAT CCAATTTC19                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GTCAGGGGCCCCAGTTTAT19                                                         (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ACAGCGTCTCTCGGGAGA18                                                      

We claim:
 1. A method of obtaining polyclonal anti-T cell antibodiesspecific to a subset of T cells having common TCR Vβ gene usagecomprising the steps of:(a) incubating T cells with an effective amountof a superantigen under conditions and for a time sufficient to allowdivision and growth of T cells reactive to the superantigen; (b)injecting the incubated T cells from step (a)into a mammal; and (c)removing blood from the mammal and isolating from said blood serumcontaining the antibodies from the mammal.
 2. The method according toclaim 1 wherein the superantigen is derived from a source selected fromthe group consisting of bacteria and retroviruses.
 3. The methodaccording to claim 2 wherein the bacterially derived superantigens areselected from the group consisting of staphylococci, mycoplasma andstreptococci.
 4. The method according to claim 3 wherein thesuperantigen is the Mycoplasma arthritidis-derived superantigen MAM. 5.A method of obtaining anti-T cell monoclonal antibodies specific to asubset of T cells having common TCR Vβ gene usage comprising the stepsof:(a) incubating T cells with an effective amount of a superantigenunder conditions and for a time sufficient to allow division and growthof T cells reactive to the superantigen; (b) injecting the incubated Tcells from step (a) into a mouse; (c) fusing splenocytes from the mousewith a plasmacytoma cell line to produce a multiplicity of hybridomas;and (d) selecting a hybridoma secreting the anti-T cell antibody fromamong those produced in step (c).
 6. The method according to claim 5wherein the superantigen is derived from a source selected from thegroup consisting of bacteria and retroviruses.
 7. The method accordingto claim 6 wherein the bacterially derived superantigens are selectedfrom the group consisting of staphylococci, mycoplasma and streptococci.8. The method according to claim 7 wherein the superantigen is theMycoplasma arthritidis-derived superantigen MAM.
 9. A method forobtaining unique polyclonal anti-T cell antibodies specific to a subsetof T cells having common TCR Vβ gene usage, comprising the stepsof:contacting T cells with pre-existing polyclonal antibodies specificto a first subset of T cells having common TCR Vβ gene usage; incubatingthe cells and antibodies for a time and under conditions sufficient toallow immune complexes to form between the antibodies and the cells;removing the immune complexes to obtain a T cell sample enriched for Tcells not recognized by the antibodies; incubating the T cell samplewith an effective amount of a superantigen under conditions and for atime sufficient to allow division and growth of T cells; injecting theresulting T cells into a mammal; and removing blood from the mammal andisolating from said blood serum containing anti-T cell antibodiesspecific to a second subset of T cells having common TCR Vβ gene usage.10. The method according to claim 9 further comprising repeating thesteps of claim 9, wherein the pre-existing antibodies comprisepolyclonal anti-T cell Vβ antibodies produced by the method of claim 9,so as to develop a panel of polyclonal anti-T cell antibodies whereineach of said antibodies recognizes a separate Vβ gene product.
 11. Amethod for obtaining unique monoclonal anti-T cell antibodies specificto a subset of T cells having common TCR Vβ gene usage, comprising thesteps of:contacting T cells with pre-existing polyclonal antibodiesspecific to a first subset of T cells having common TCR Vβ gene usage;incubating the cells and antibodies for a time and under conditionssufficient to allow immune complexes to form between the antibodies andthe cells; removing the immune complexes to obtain a T cell sampleenriched for T cells not recognized by the antibodies; incubating the Tcell sample with an effective amount of a superantigen under conditionsand for a time sufficient to allow division an d growth of T cells;injecting the resulting T cells into a mouse; fusing splenocytes fromthe mouse with a plasmacytoma cell line to produce a multiplicity ofhybridomas; and selecting a hybridoma secreting anti-T cell antibodiesspecific to a second subset of T cells having common TCR Vβ gene usagefrom among those produced in the previous step.
 12. The method accordingto claim 11 further comprising repeating the steps of claim 11, whereinthe pre-existing antibodies comprise polyclonal anti-T cell Vβantibodies produced by the methods of claims 9 or 10, so as to develop apanel of monoclonal anti-T cell antibodies wherein each of saidantibodies recognizes a separate Vβ gene product.
 13. A method forobtaining unique monoclonal anti-T cell antibodies specific to a subsetof T cells having common TCR Vβ gene usage, comprising the stepsof:contacting T cells with pre-existing monoclonal antibodies specificto a first subset of T cells having common TCR Vβ gene usage; incubatingthe cells and antibodies for a time and under conditions sufficient toallow immune complexes to form between the antibodies and the cells;removing the immune complexes to obtain a T cell sample enriched for Tcells not recognized by the antibodies; incubating the T cell samplewith an effective amount of a superantigen under conditions and for atime sufficient to allow division and growth of T cells; injecting theresulting T cells into a mouse; fusing splenocytes from the mouse with aplasmacytoma cell line to produce a multiplicity of hybridomas; andselecting a hybridoma secreting anti-T cell antibodies specific to asecond subset of T cells having common TCR Vβ gene usage from amongthose produced in the previous step.
 14. The method according to claim13 further comprising repeating the steps of claim 13, wherein thepre-existing antibodies comprise monoclonal anti-T cell Vβ antibodiesproduced by the method of claim 13, so as to develop a panel of anti-Tcell antibodies wherein each of said antibodies recognizes a separate Vβgene product.